ABSTRACT/PROJECT SUMMARY In general, the lymphatic system plays a profound role in maintaining health by regulating tissue fluid homeostasis, immune surveillance, blood pressure and reverse cholesterol transport. Lymphatic abnormalities are associated with lymphedema, fibrosis, tumor metastasis, obesity and sepsis. The pulmonary lymphatic system is especially important for neonatal survival by clearing lung fluids at the birth transition to air breathing as well as throughout adult life. This underscores how essential pulmonary lymphatics have been to mammalian evolution. Furthermore, the clinical relevance of pulmonary lymphatics is apparent based on their contributions to lung infections and diseases such as bronchopulmonary dysplasia, tuberculosis, asthma, cancer, lymphangioleiomyomatosis, allograft rejection, sarcoidosis, and interstitial pulmonary fibrosis. Despite their fundamental importance, there is little information regarding the precise molecular and cellular mechanisms that lead to pulmonary lymphatic establishment and maturation. In addition, virtually nothing is known regarding lymphatic regeneration during tissue repair in the adult lung. Based on our preliminary data, our working hypothesis is that during embryonic development pulmonary lymphatic endothelial cells (LECs) arise from diverse origins, which coalesce and mature to ensure lung fluid clearance, air breathing and ultimately neonatal survival. We further hypothesize that LEC regeneration occurs through cell-cycle reentry and reactivation of lymphatic developmental signals during adult lung repair. Modern lineage tracing techniques have supported Florence Sabin's 100-year old model of lymphatic development, which states that all LECs are derived from the embryonic veins. Interestingly, two of our in-vivo lineage-tracing experiments suggest that a subset of pulmonary LECs is derived from a non- venous origin, consistent with recent discoveries in other organs. In Specific Aim 1, we will identify the precise cellular origins of LECs in the embryonic lung. Prior to birth, functional maturation of diverse lung cell types must occur to ensure fetal lung fluid clearance, respiration and neonatal survival. For Specific Aim 2, we will determine the mechanisms of LEC maturation, for which we have developed and optimized a robust flow-cytometry method to isolate highly enriched LECs from embryonic and adult lungs. In Specific Aim 3, we focus on the adult lung to test the hypothesis that LEC regeneration occurs and that it involves cell cycle reentry and reactivation of lymphatic developmental signals in a pneumonectomy (PNX) mouse model of adult lung tissue repair. Results of these studies will enhance our understanding of fundamental aspects concerning lung and lymphatic biology across the entire life span. It is hoped that a better understanding of pulmonary lymphatic development will reveal potential therapeutic targets to enhance lymphatic function or to augment or inhibit lymph-angiogenesis in pathologic conditions that affect the lung.